Field of the Invention
The invention relates to a nucleotide sequence, a universal reverse primer, a universal RT primer, a method for designing primer, and a miRNA detection method, and more particularly, to a nucleotide sequence, a universal reverse primer, a universal RT primer, a method for designing primer, and a miRNA detection method for miRNA detection, quantification, and expression profiling.
Description of Related Art
miRNAs are highly conserved non-coding RNAs having a length between 18 to 25 nucleotides, and regulate gene expression of an organism. In recent years, research results have shown that miRNA expression is implicated in many diseases, especially cancer, and miRNAs function as oncogenes or tumor suppressor genes which play important roles in pathophysiology of human cancer. More specifically, the level/expression of miRNA in cancer patients' bodily fluids can be used as a tool for cancer diagnosis and prognosis, which makes miRNAs novel, robust cancer biomarkers. Moreover, various cancer diagnostic tools have been developed based on the difference in miRNA expression between healthy individuals and cancer patients. Therefore, the materials and methods used for miRNA detection, quantification, and expression profiling are important and critical.
In prior art, methods of detecting and quantifying miRNA include Northern blot hybridization, cloning, microarray gene chip analysis, and next-generation sequencing technology. In comparison to prior art, the quantitative real-time reverse transcription PCR (qRT-PCT) method has higher sensitivity and specificity.
Currently, various different qRT-PCR methods have been developed, and these methods can mainly be divided into the following two types. The first type is performing cDNA synthesis using a stem-loop RT primer, and quantifying miRNA using a miRNA specific probe or a universal probe. The second method is to perform cDNA synthesis using a linear universal RT primer and quantify miRNA using a miRNA specific forward primer, a RT-primer specific reverse primer, and a double-stranded DNA intercalating dye.
Regarding the qRT-PCR method, since the reverse transcription primer of the first method contains a one stem-loop structure, better specificity is achieved, but the cost of the miRNA specific probe or the universal probe used is higher. The second method does not require a probe, and therefore costs can be further reduced. However, the specificity of the linear universal RT primer used in the second method is worse than that of the stem-loop reverse transcription primer of the first type, and the current commercial universal RT primer may need to be further modified.
Based on the above, how to increase the specificity and the sensitivity of the linear universal RT primer and the reverse primer and achieving the characteristics of reduced cost and ease of use so as to detect, quantify, and analyze the performance of miRNA is an important topic.